There are currently 5000 species of Aphididae with over 100 species economically important as pests of crops (Blackman & Eastop 2006, 2007). Aphids are phloem sap feeders that damage the plants through the removal of carbohydrates and amino acids and by injecting phytotoxic saliva and vectoring plant diseases while feeding. The Russian wheat aphid, Diuraphis noxia, and the greenbug, Schizaphis graminum, are globally distributed and significant pests of cereals, causing losses exceeding $250 million/year in the mid-western United States alone (Webster 1994, Morrison and Peairs 1998). M. persicae attacks over 200 species of plants and is known by many names including green peach aphid or peach-potato aphid. Plant damage occurs mainly from the aphid's transmission of viruses lethal to many vegetables and tobacco (Eastop 1977. Blackman and Eastop 2000). The low profit margins in crop production and frequent insecticide use required to mitigate losses caused by aphids are economically unsustainable.
Chemical pesticides such as pyrethrins and pyrethroids are the most common means of controlling aphids. However the use of traditional chemical pesticides has disadvantages, including non-target effects on neutral or beneficial insects, as well as other animals. Chemical pesticide usage also can lead to chemical residue run-off into streams and seepage into water supplies resulting in ecosystem/environment damage. In addition, animals higher in the food chain are at risk when they consume pesticide contaminated crops or insects. The handling and application of chemical pesticides also presents exposure danger to the public and professionals, and could lead to accidental dispersal into unintended environmentally sensitive areas. In addition, prolonged chemical pesticide application may result in an insect population becoming resistant to a chemical pesticide. In order to control a traditionally chemical resistant-pest, new more potent chemical pesticides must be utilized, which in turn will lead to another resistance cycle. As such, there is a need in the art to control pest populations without the disadvantages of traditional chemical pesticides.
An approach to decrease dependence on chemical pesticides is by causing a specific gene(s) of the target-pest to malfunction by either over expression or silencing gene expression. The silencing approach utilizes RNA interference pathways to knockdown the gene of interest via double stranded RNA. Double stranded RNA (dsRNA) induces sequence—specific post-transcriptional gene silencing in many organisms by a process known as RNA interference (RNAi). RNAi is a post-transcriptional, highly conserved process in eukaryotes that leads to specific gene silencing through degradation of the target mRNA. The silencing mechanism is mediated by dsRNA that is homologous in sequence to the gene of interest. The dsRNA is processed into small interfering RNA (siRNA) by an endogenous enzyme called DICER inside the target pest, and the siRNAs are then incorporated into a multi-component RNA-induced silencing complex (RISC), which finds and cleaves the target mRNA. The dsRNA inhibits expression of at least one gene within the target, which exerts a deleterious effect upon the target.
Fire, et al. (U.S. Pat. No. 6,506,559) discloses a process of introducing RNA into a living cell to inhibit gene expression of a target gene in that cell. The RNA has a region with double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequences of the duplex region of the RNA and of a portion of the target gene are identical. Specifically, Fire, et al. (U.S. Pat. No. 6,506,559) discloses a method to inhibit expression of a target gene in a cell, the method comprising introduction of a double stranded ribonucleic acid into the cell in an amount sufficient to inhibit expression of the target gene, wherein the RNA is a double-stranded molecule with a first ribonucleic acid strand consisting essentially of a ribonucleotide sequence which corresponds to a nucleotide sequence of the target gene and a second ribonucleic acid strand consisting essentially of a ribonucleotide sequence which is complementary to the nucleotide sequence of the target gene. Furthermore, the first and the second ribonucleotide strands are separately complementary strands that hybridize to each other to form the said double-stranded construct, and the double-stranded construct inhibits expression of the target gene.
To utilize RNA interference as a method to regulate gene expression to control a target organism, a specific essential gene needs to be targeted. One such gene is the Chloride Intracellular Channel (CLIC) gene. The CLIC gene encodes a multifunctional protein thought to be involved in number of cellular processes based on its role in glutathione signaling and in allowing chloride ion flux across membranes (Averaimo, et al. 2010).
Another gene of interest is Sucrase. The Sucrase gene encodes a protein responsible for the hydrolysis of sucrose into fructose and glucose. Sucrose is the main plant sugar, and so is the most important sustenance for plant-feeding insects. Interference with an insect's ability to metabolize sucrose will thereby starve the insect (Karley, et al. 2005).
Such novel control methods that would induce silencing of CLIC and Sucrase would be desirable as they avoid the undesirable characteristics of traditional chemical pesticides. Traditional chemical pesticides in general have the disadvantage of being toxic to the environment as well as affecting a broad range of insect. To that end, there is a need to develop dsRNA constructs that are engineered to target and silence CLIC and Sucrase mRNA that would overcome some of the disadvantages of using traditional pesticides and that can target specific pests.